Rapid immunoprecipitation mass spectrometry of endogenous proteins

RIME was performed as previously described (41) except that cortical neurons at DIV 7 were subject to the ChIP procedure described above. Precipitated protein complexes were boiled for 10 min in SDS sample buffer and separated by SDS–polyacrylamide gel electrophoresis (PAGE). Gel bands were digested overnight with trypsin (Pierce), followed by destaining, reduction with dithiothreitol, and alkylation with iodoacetamide (Sigma-Aldrich). The samples then underwent solid-phase extraction cleanup with an Oasis HLB plate (Waters), and the resulting samples were injected onto an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLCnano liquid chromatography system. Samples were injected onto a 75-μm i.d., 75-cm long EasySpray column (Thermo Fisher Scientific) and eluted with a gradient from 0 to 28% buffer B over 90 min. Buffer A contained 2% (v/v) acetonitrile (ACN) and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 1.8 kV and an ion transfer tube temperature of 275°C. MS scans were acquired at 120,000 resolution in the Orbitrap, and up to 10 tandem MS (MS/MS) spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation for ions with charges 2 to 7. Dynamic exclusion was set for 20 s after an ion was selected for fragmentation.

Raw MS data files were analyzed using Proteome Discoverer v2.2 (Thermo Fisher Scientific), with peptide identification using Sequest HT searching against the mouse protein database from UniProt. Fragment and precursor tolerances of 10 parts per million and 0.6 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys was set as a fixed modification, with oxidation of Met set as a variable modification. Five percent of FDR cutoff was used to determine enriched polypeptides. Immunoglobulin G IP control was used to exclude nonspecifically associated polypeptides. PSMs indicate the number of peptide spectrum matches or the number of spectra assigned to peptides that contributed to the inference of the protein. Abundance indicates the sum of the peak intensities for each peptide identified for that protein.